Oxygen carriers that are useful as oxygen therapeutics (sometimes referred to as “oxygen-carrying plasma expanders”) can be grouped into the following three categories: i) perfluorocarbon-based emulsions, ii) liposome-encapsulated Hb and iii) modified Hb. As discussed below, none has been entirely successful, though products comprising modified cell-free Hb are thought to be the most promising. Perfluorochemical-based emulsions dissolve oxygen as opposed to binding it as a ligand. In order to be used in biological systems, the perfluorochemical must be emulsified with a lipid, typically egg-yolk phospholipid. Though the perfluorocarbon emulsions are inexpensive to manufacture, they do not carry sufficient oxygen at clinically tolerated doses to be effective. Conversely, while liposome-encapsulated Hb has been shown to be effective, it is too costly for widespread use. (See generally, Winslow, R. M., “Hemoglobin-based Red Cell Substitutes,” Johns Hopkins University Press, Baltimore (1992)).
Initial attempts to utilize free Hb from erythrocyte hemolysates as a red cell substitute were unsuccessful. The stromal components were found to be toxic, resulting in coagulopathy and associated renal failure. In 1967, stroma-free Hb (“SFH”) solutions had been prepared (Rabiner, S. F. et al., 1967, J. Exp. Med. 126:1127-1142). However, they were found to have a transfusion half-life of only about 100 minutes.
The reason for the short circulation half-life of SFH is due to the ability of the protein to dissociate from its tetrameric form into dimers, which are rapidly filtered from the circulation by the kidneys. Accordingly, a multitude of methods for cross-linking Hb, and other means for increasing the hydrodynamic size of Hb by conjugation with macromolecules, have been devised to limit or prevent the extravasation of Hb. Cross-linking SFH to form poly-Hb is described in U.S. Pat. Nos. 4,001,200 and 4,001,401. Internally cross-linked Hb, which binds amino acid residues between subunits, may be achieved with diaspirin (diesters of bis-3,5-dibromosaliocylate) as described in U.S. Pat. No. 4,529,719) or 2-N-2-formyl-pyridoxal-5′-phosphate and borohydride (Benesch, R. E. et al, 1975, Biochem. Biophys. Res. Commun. 62:1123-1129). Intramolecular cross-linking, which chemically binds subunits of the tetrameric Hb unit to prevent the formation of dimers, is disclosed in U.S. Pat. No. 5,296,465. In addition, Simon, S. R. and Konigsberg, W. H. disclosed the use of bis-(N-maleimidomethyl) ether (“BME”) to generate intramolecularly cross-linked Hb (1966, PNAS 56:749-56) that was reported to have a four fold increase in half-life when infused into rats and dogs (Bunn, H. F. et al., 1969, J. Exp. Med. 129:909-24). However, the cross-linking of Hb with BME resulted in the concomitant increase in the oxygen affinity of Hb, which at the time was thought to prevent its use as a potential Hb-based oxygen carrier (“HBOC”).
SFH was also linked to other macromolecules such as dextran (Chang, J. E. et al., 1977, Can. J. Biochem. 55:398-403), hydroxyethyl starch (DE 2,161,086), gelatin (DE 2,449,885), albumin (DE 2,449,885) and PEG (DE 3,026,398, U.S. Pat. Nos. 4,670,417, 4,412,989 and 4,301,144).
Some of the physiological effects of these oxygen carrying solutions are not fully understood. Of these, perhaps the most controversial is the propensity to cause vasoconstriction, which may manifest as hypertension in animals and man (Amberson, W., 1947, Science 106:117-117) (Keipert, P. et al., 1993, Transfusion 33:701-708). Human Hb cross-linked between α-chains with bis-dibromosalicyl-fumarate (“ααHb”) was developed by the U.S. Army as a model red cell substitute, but was abandoned after it showed severe increases in pulmonary and systemic vascular resistance (Hess, J. et al., 1991, Blood 78:356A). A commercial version of this product was also abandoned after a disappointing Phase III clinical trial (Winslow, R. M., 2000, Vox Sang 79:1-20).
The most common explanation for the vasoconstriction produced by cell-free Hb is that it readily binds the endothelium-derived relaxing factor (EDRF), nitric oxide (“NO”). Two molecular approaches have been advanced in attempting to overcome the NO binding activity of Hb. The first approach was utilizing recombinant DNA, which attempted to reduce the NO binding of Hb by site-specific mutagenesis of the distal heme pocket (Eich, R. F. et al., 1996, Biochem. 35:6976-83). The second approach utilized chemical modification in which the size of the Hb was enhanced through oligomerization, which attempted to reduce or possibly completely inhibit the extravasation of Hb from the vascular space into the interstitial space (Hess, J. R. et al., 1978, J. Appl. Physiol. 74:1769-78; Muldoon, S. M. et al., 1996, J. Lab. Clin. Med. 128:579-83; Macdonal, V. W. et al., 1994, Biotechnology 22:565-75; Furchgott, R., 1984, Ann. Rev. Pharmacol. 24:175-97; and Kilbourne, R. et al., 1994, Biochem. Biophys. Res. Commun. 199:155-62).
In fact, recombinant Hbs with reduced affinity for NO have been produced that are less hypertensive in top-load rat experiments (Doherty, D. H. etg al. 1998, Nature Biotechnology 16:672-676 and Lemon, D. D. et al. 1996, Biotech 24:378). However, studies suggest that NO binding may not be the only explanation for the vasoactivity of Hb. It has been found that certain large Hb molecules, such as those modified with PEG, were virtually free of the hypertensive effect, even though their NO binding rates were identical to those of the severely hypertensive ααHb (Rohlfs, R. J. et al. 1998, J Biol. Chem. 273:12128-12134). Furthermore, it was found that PEG-Hb was extraordinarily effective in preventing the consequences of hemorrhage when given as an exchange transfusion prior to hemorrhage (Winslow, R. M. et al. 1998, J. Appl. Physiol. 85:993-1003).
The conjugation of PEG to Hb reduces its antigenicity and extends its circulation half-life. However, the PEG conjugation reaction has been reported to result in dissociation of Hb tetramers into αβ-dimer subunits causing gross hemoglobinuria in exchange-transfused rats receiving PEG-conjugates of Hb monomeric units below 40,000 Daltons (“Da”) (Iwashita and Ajisaka Organ-Directed Toxicity: Chem. Indicies Mech., Proc. Symp., Brown et al. 1981, Eds. Pergamon, Oxford, England pgs 97-101). A polyalkylene oxide (“PAO”) conjugated Hb having a molecular weight greater than 84,000 Da was prepared by Enzon, Inc. (U.S. Pat. No. 5,650,388) that carried 10 copies of PEG-5,000 chains linked to Hb at its α and ε-amino groups. This degree of substitution was described as avoiding clinically significant nephrotoxicity associated with hemoglobinuria in mammals. However, the conjugation reaction resulted in a heterogeneous conjugate population and contained other undesirable reactants that had to be removed by column chromatography.
PEG conjugation is typically carried out through the reaction of an activated PEG with a functional group on the surface of biomolecules. The most common functional groups are the amino groups of lysine and histidine residues, and the N-terminus of proteins; thiol groups of cysteine residues; and the hydroxyl groups of serine, threonine and tyrosine residues and the C-terminus of the protein. PEG is usually activated by converting the hydroxyl terminus to a reactive moiety capable of reacting with these functional groups in a mild aqueous environment. One of the most common monofunctional PEGs used for conjugation of therapeutic biopharmaceuticals is methoxy-PEG (“mPEG”), which has only one functional group (i.e. hydroxyl), thus minimizing cross-linking and aggregation problems that are associated with bifunctional PEG. However, mPEG is often contaminated with high molecular weight bifunctional PEG (i.e. “PEG diol”), which can range as high as 10 to 15% (Dust J. M. et al. 1990, Macromolecule 23:3742-3746), due to its production process. This bifunctional PEG diol has roughly twice the size of the desired monofunctional PEG. The contamination problem is further aggravated as the molecular weight of PEG increases. The purity of mPEG is especially critical for the production of PEGylated biotherapeutics, because the FDA requires a high level of reproducibility in the production processes and quality of the final drug product.
Conjugation of Hb to PAOs has been performed in both the oxygenated and deoxygenated states. U.S. Pat. No. 6,844,317 describes conjugating Hb in the oxygenated, or “R” state, to enhance the oxygen affinity of the resultant PEG-Hb conjugate. This is accomplished by equilibrating Hb with the atmosphere prior to conjugation. Others describe a deoxygenation step prior to conjugation to diminish the oxygen affinity and increase structural stability enabling the Hb to withstand the physical stresses of chemical modification, diafiltration and/or sterile filtration and sterilization (U.S. Pat. No. 5,234,903). For intramolecular cross-linking of Hb, it is suggested that deoxygenating Hb prior to modification may be required to expose lysine 99, of the α-chain, to the cross-linking reagent (U.S. Pat. No. 5,234,903).
The kinetics of Hb thiolation with iminothiolane prior to conjugation with PEG was investigated by Acharya et al. (U.S. Pat. No. 7,501,499). It was observed that increasing the concentration of iminothiolane from 10-fold, which introduced an average of five extrinsic thiols per tetramer, to 30-fold nearly doubled the number of extrinsic thiols on Hb. However, the size enhancement seen after PEG conjugation was only marginal, even with double the number of thiols. This suggested that the conjugation reaction in the presence of 20-fold molar excess of maleimidyl PEG-5000 covered the surface of the Hb with less reactive thiols resulting in steric interference that resisted further modification of Hb with more reactive thiols. Consequently, to achieve the desired molecular weight of modified Hb (i.e. 6±1 PEG per Hb molecule), Acharya et al. thiolated Hb with an 8-15 molar excess of iminothiolane, and then reacted the thiolated Hb with a 16-30 fold molar excess of maleimidyl PEG-5000. However, these high molar excess reactant concentrations in large scale production significantly increase the cost for preparing the HBOC. Moreover, such high molar excess of the maleimidyl PEG-5000 results in a more heterogeneous product with the production of a greater number of unwanted reactants.
Accordingly, there is a need for a method of preparing PEG conjugated Hb of a particular size range with decreased cost, increased efficiency, less impurities, and narrower molecular weight range.